title: Distinct specificity and single-molecule kinetics characterize the interaction of pathogenic and non-pathogenic antibodies against platelet factor 4-heparin complexes with platelet factor 4.
abstract: Heparin-induced thrombocytopenia (HIT) is a thrombotic complication of heparin therapy mediated by antibodies to complexes between platelet factor 4 (PF4) and heparin or cellular glycosaminoglycans. However, only a fraction of patients with anti-PF4-heparin antibodies develop HIT, implying that only a subset of these antibodies is pathogenic. The basis for the pathogenic potential of anti-PF4-heparin antibodies remains unclear. To elucidate the intrinsic PF4-binding properties of HIT-like monoclonal antibody (KKO) versus non-pathogenic antibody (RTO) at the single-molecule level, we utilized optical trap-based force spectroscopy to measure the strength and probability of binding of surface-attached antibodies with oligomeric PF4 to simulate interactions on cells. To mimic the effect of heparin in bringing PF4 complexes into proximity, we chemically cross-linked PF4 tetramers using glutaraldehyde. Analysis of the force histograms revealed that KKO-PF4 interactions had ∼10-fold faster on-rates than RTO-PF4, and apparent equilibrium dissociation constants differed ∼10-fold with similar force-free off-rates (k(off) = 0.0031 and 0.0029 s(-1)). Qualitatively similar results were obtained for KKO and RTO interacting with PF4-heparin complexes. In contrast to WT PF4, KKO and RTO showed lower and similar binding probabilities to cross-linked PF4(K50E), which forms few if any oligomers. Thus, formation of stable PF4 polymers results in much stronger interactions with the pathogenic antibody without a significant effect on the binding of the non-pathogenic antibody. These results suggest a fundamental difference in the antigen-binding mechanisms between model pathogenic and non-pathogenic anti-PF4 antibodies that might underlie their distinct pathophysiological behaviors.
authors.affiliation: From the Department of Cell and Developmental Biology and.
authors.forename: Rustem I
authors.initials: RI
authors.lastname: Litvinov
authors.forename: Serge V
authors.initials: SV
authors.lastname: Yarovoi
authors.forename: Lubica
authors.initials: L
authors.lastname: Rauova
authors.forename: Valeri
authors.initials: V
authors.lastname: Barsegov
authors.forename: Bruce S
authors.initials: BS
authors.lastname: Sachais
authors.forename: Ann H
authors.initials: AH
authors.lastname: Rux
authors.forename: Jillian L
authors.initials: JL
authors.lastname: Hinds
authors.forename: Gowthami M
authors.initials: GM
authors.lastname: Arepally
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Cines
authors.forename: John W
authors.initials: JW
authors.lastname: Weisel
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000918:Antibody Specificity; D000925:Anticoagulants / Q000009:adverse effects / Q000737:chemistry* / Q000276:immunology / Q000627:therapeutic use; D001323:Autoantibodies / Q000737:chemistry* / Q000276:immunology; D001666:Binding Sites, Antibody; D006493:Heparin / Q000009:adverse effects / Q000737:chemistry* / Q000276:immunology / Q000627:therapeutic use; D006801:Humans; D007700:Kinetics; D010978:Platelet Factor 4 / Q000737:chemistry* / Q000276:immunology; D013921:Thrombocytopenia / Q000139:chemically induced / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000925:Anticoagulants; D001323:Autoantibodies; D010978:Platelet Factor 4; D006493:Heparin
keywords: Antibodies; Heparin-binding Protein; Platelets; Single Molecule Biophysics; Thrombosis